Energy conversion, utilization, and access underlie many of the great challenges of our time, including those associated with sustainability, environmental quality, security, and poverty. New applications of emerging technologies are required to respond to these challenges. Biotechnology, one of the most powerful of the emerging technologies, can give rise to important new energy conversion processes. Plant biomass and derivatives thereof are a resource for the biological conversion of energy to forms useful to humanity.
Biomass is from living, or recently living organisms, such as wood, waste, (hydrogen) gas, and alcohol fuels. Biomass is carbon, hydrogen, and oxygen based. Nitrogen and small quantities of other atoms, including alkali, alkaline earth and heavy metals can be found as well. Metals are often found in functional molecules such as the porphyrins which include chlorophyll which contains magnesium. Plants in particular combine water and carbon dioxide to sugar building blocks. The required energy is produced from light via photosynthesis based on chlorophyll. On average, between 0.1% and 1% of the available light is stored as chemical energy in plants. The sugar building blocks are the starting point for all of the major fractions found in terrestrial plants, lignin, hemicellulose, and cellulose. Biomass is widely recognized as a promising source of raw material for production of renewable fuels and chemicals. The primary obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost technology for overcoming the recalcitrance of these materials to conversion into useful fuels. Biomass contains carbohydrate fractions (e.g., starch, cellulose, and hemicellulose) that can be converted into ethanol. In order to convert these fractions, the starch, cellulose, and hemicellulose must ultimately be converted or hydrolyzed into monosaccharides; it is the hydrolysis that has historically proven to be problematic.
Biologically mediated processes are promising for energy conversion, in particular, for the conversion of biomass into fuels. Biomass processing schemes involving enzymatic or microbial hydrolysis commonly involve four biologically mediated transformations: (1) the production of saccharolytic enzymes (amylases, cellulases, and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). These four transformations occur in a single step in a process configuration called consolidated bioprocessing (CBP), which is distinguished from other less highly integrated configurations in that it does not involve a dedicated process step for cellulase and/or hemicellulase production.
CBP offers the potential for lower cost and higher efficiency than processes featuring dedicated saccharolytic enzyme production. The benefits result in part from avoided capital costs, substrate, and other raw materials, and utilities associated with saccharolytic enzyme production. In addition, several factors support the realization of higher rates of hydrolysis, and hence reduced reactor volume and capital investment using CBP, including enzyme-microbe synergy and the use of thermophilic organisms and/or complexed saccharolytic systems. Moreover, cellulose-adherent cellulolytic microorganisms are likely to compete successfully for products of cellulose hydrolysis with non-adhered microbes, e.g., contaminants, which could increase the stability of industrial processes based on microbial cellulose utilization. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring saccharolytic microorganisms to improve product-related properties, such as yield and titer; and engineering non-saccharolytic organisms that exhibit high product yields and titers to express a heterologous saccharolytic enzyme system enabling starch, cellulose, and hemicellulose utilization.
The breakdown of starch down into sugar requires amylolytic enzymes. Amylase is an example of an amylolytic enzyme that is present in human saliva, where it begins the chemical process of digestion. The pancreas also makes amylase (alpha amylase) to hydrolyze dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce amylases. Amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds.
Several amylolytic enzymes are implicated in starch hydrolysis. Alpha-amylases (EC 3.2.1.1) (alternate names: 1,4-α-D-glucan glucanohydrolase; glycogenase) are calcium metalloenzymes, i.e., completely unable to function in the absence of calcium. By acting at random locations along the starch chain, alpha-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and “limit dextrin” from amylopectin. Because it can act anywhere on the substrate, alpha-amylase tends to be faster-acting than beta-amylase. Another form of amylase, beta-amylase (EC 3.2.1.2) (alternate names: 1,4-α-D-glucan maltohydrolase; glycogenase; saccharogen amylase) catalyzes the hydrolysis of the second α-1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. The third amylase is gamma-amylase (EC 3.2.1.3) (alternate names: Glucan 1,4-α-glucosidase; amyloglucosidase; Exo-1,4-α-glucosidase; glucoamylase; lysosomal α-glucosidase; 1,4-α-D-glucan glucohydrolase). In addition to cleaving the last α(1-4)glycosidic linkages at the nonreducing end of amylose and amylopectin, yielding glucose, gamma-amylase will cleave α(1-6) glycosidic linkages.
A fourth enzyme, alpha-glucosidase, acts on maltose and other short malto-oligosaccharides produced by alpha-, beta-, and gamma-amylases, converting them to glucose.
Three major types of enzymatic activities are required for native cellulose degradation. The first type are endoglucanases (1,4-β-D-glucan 4-glucanohydrolases; EC 3.2.1.4). Endoglucanases cut at random in the cellulose polysaccharide chain of amorphous cellulose, generating oligosaccharides of varying lengths and consequently new chain ends. The second type are exoglucanases, including cellodextrinases (1,4-β-D-glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases (1,4-β-D-glucan cellobiohydrolases; EC 3.2.1.91). Exoglucanases act in a processive manner on the reducing or non-reducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Exoglucanases also act on microcrystalline cellulose, presumably peeling cellulose chains from the microcrystalline structure. The third type are β-glucosidases (β glucoside glucohydrolases; EC 3.2.1.21). β-glucosidases hydrolyze soluble cellodextrins and cellobiose to glucose units.
A variety of plant biomass resources are available as starch and lignocellulosics for the production of biofuels, notably bioethanol. The major sources of plant biomass resources are (i) wood residues from paper mills, sawmills, and furniture manufacturing, (ii) municipal solid wastes, (iii) agricultural residues, and (iv) energy crops such as corn. Pre-conversion of particularly the cellulosic fraction in these biomass resources (using physical, chemical, or enzymatic processes) to fermentable sugars (glucose, cellobiose, maltose, alpha- and cellodextrins) would enable their fermentation to bioethanol, provided the necessary fermentative micro-organism with the ability to utilize these sugars is used.
On a world-wide basis, 1.3×1010 metric tons (dry weight) of terrestrial plants are produced annually (Demain, A. L., et al., Microbiol. Mol. Biol. Rev. 69:124-154 (2005)). Plant biomass consists of about 40%-55% cellulose, 25%-50% hemicellulose and 10%-40% lignin, depending whether the source is hardwood, softwood, or grasses (Sun, Y. and Cheng, J., Bioresource Technol. 83:1-11 (2002)). The major polysaccharide present is water-insoluble, cellulose that contains the major fraction of fermentable sugars (glucose, cellobiose or cellodextrins).
Hemicellulose oligomers represent a significant portion of lignocellulosic feedstocks. In hardwood species, carbohydrate structures with monomeric components including xylose, mannose, galactose, and arabinose make up as much as 20% of the feedstock by weight. Several methods of biomass pretreatment produce a mixture of soluble oligomers and monomers, including xylo-oligomers and gluco-oligomers in addition to those cited above. In addition, an insoluble fraction containing glucan, additional hemicellulose oligomers, and lignin is produced. Aqueous pretreatments in particular leave hemicellulose oligomers intact, and the conversion of this mixture of soluble oligomers is achieved using acid hydrolysis (Kim, Y., Kreke, T., Ladisch, M. R. Reaction mechanisms and kinetics of xylo-oligosaccharide hydrolysis by dicarboxylic acids. AICHe Journal. (2012). Article first published online: 23 Apr. 2012) or enzymatic hydrolysis prior to fermentation, with varying degrees of efficiency and cost. Acid hydrolysis in particular requires increased costs due to reaction vessels that require the ability to withstand low pH, high temperature, and pressure, although high yields have been reported (Kim, Y., Kreke, T., Ladisch, M. R. Reaction mechanisms and kinetics of xylo-oligosaccharide hydrolysis by dicarboxylic acids. AICHe Journal. (2012). Article first published online: 23 Apr. 2012). In addition, it is known that the hydrolysis of xylo-oligomers is very important for improving the kinetics of cellulose hydrolysis by cellulase as these enzymes are very inhibited by these oligomers (Qing, Q., Yang, B., Wyman, C. E. Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes. Bioresource Technol. 101:9624-9630 (2010); see also U.S. application Ser. No. 13/055,366, published as U.S. Pub. No. 2011/0201084). As shown below, several commercially available enzyme preparations are relatively poor at achieving high yield enzymatic hydrolysis of substituted, soluble oligomers derived from hardwood.
Bakers' yeast (Saccharomyces cerevisiae) remains the preferred micro-organism for the production of ethanol (Hahn-Hagerdal, B., et al., Adv. Biochem. Eng. Biotechnol. 73:53-84 (2001)). Attributes in favor of this microbe are (i) high productivity at close to theoretical yields (0.51 g ethanol produced/g glucose used), (ii) high osmo- and ethanol tolerance, (iii) natural robustness in industrial processes, (iv) being generally regarded as safe (GRAS) due to its long association with wine and bread making, and beer brewing. Furthermore, S. cerevisiae exhibits tolerance to inhibitors commonly found in hydrolyzates resulting from biomass pretreatment. The major shortcoming of S. cerevisiae is its inability to utilize complex polysaccharides such as starch, cellulose, and polymeric hemicellulose or its break-down products, such as cellobiose, xylose, and cellodextrins.
As noted above, ethanol producing yeast such as S. cerevisiae require addition of external cellulases when cultivated on cellulosic substrates such as pre-treated wood because this yeast does not produce endogenous cellulases. Functional expression of fungal cellulases such as T. reesei CBH1 and CBH2 in yeast S. cerevisiae have been demonstrated (Den Haan R et al., Metab. Eng., 9:87-94 (2007)). However, current levels of expression and specific activity of cellulases heterologously expressed in yeast are still not maximally efficient with respect to the lignocellulosic substrate. Thus, there remains a significant need for improvement in the amount and variety of cellulase activity expressed in order to attain the goal of achieving a consolidated bioprocessing (CBP) system capable of efficiently and cost-effectively converting cellulosic substrates to ethanol.
The composition of lignocellulosic material varies greatly based on its species of origin, the particular tissue from which it is derived, and its pretreatment. Because of its varied composition, organisms designed for CBP must produce digestive enzymes that can accommodate a variety of substrates, in a variety of conformations, in a variety of reaction environments. To date, efficient usage of lignocellulosic substrates requires the addition of external enzymes at high levels. However, externally added enzymes are costly. Therefore, it would be very beneficial to isolate cellulases from cellulolytic organisms with high specific activity and high expression levels in host organisms, such as the yeast S. cerevisiae in order to achieve CBP. Also, in order to use lignocellulosic material with maximal efficiency, it would also be beneficial to discover combinations of paralogous and/or orthologous enzymes that work synergistically to achieve more efficient break down of lignocellulosic components.
Beyond fungi, there are a large variety of cellulolytic bacteria that can be used as gene donors for expression of lignocellulolytic enzymes in yeast. In one aspect, the present invention is drawn to identifying cellulolytic enzymes from a variety of organisms and subsequently identifying enzymes that work in maximally efficient combinations to digest lignocellulosic material. Given the diversity of cellulolytic bacteria, classification of these organisms based on several parameters (Lynd, L. R., et al., Microbial Cellulose Utilization: Fundamentals and Biotechnology. Microbiol. Mol. Biol. Rev., 66:506-577 (2002)) can inform the choice of gene donors. The following are distinguishing characteristics: (A) aerobic vs. anaerobic, (B) mesophiles vs. thermophiles; and (C) noncomplexed, cell free enzymes vs. complexed, cell bound enzymes.
Another consideration when defining the needed set of enzymatic activities is to attempt to characterize the linkages in a lignocellulosic substrate. FIGS. 1A-1D provide an overview of the carbohydrate structures present in plant material given in Van Zyl, W. H., et al., Consolidated bioprocessing for bioethanol production using Saccharomyces cerevisiae, Adv. Biochem. Eng. Biotechnol., 108:205-235 (2007). Intl Pub. No. WO2011/153516, which is herein incorporated by reference, provides an analysis of hardwood substrate.